Hinchcliffe E H, Linck R W
Department of Cell Biology, University of Minnesota, School of Medicine, Minneapolis, Minnesota 55455, USA.
J Cell Sci. 1998 Mar;111 ( Pt 5):585-95. doi: 10.1242/jcs.111.5.585.
Biochemical fractionation of axonemal microtubules yields the protofilament ribbon (pf-ribbon), an insoluble structure of 3-4 longitudinal protofilaments composed primarily of alpha/beta tubulin, tektins A, B and C, and two previously uncharacterized polypeptides of 77 kDa and 83 kDa. We have isolated the 77/83 kDa polypeptides (termed Sp77 and Sp83) from sperm flagella of the sea urchin Stronglyocentrotus purpuratus and raised polyclonal antibodies against them. Sp77 and Sp83 copurify exclusively with the pf-ribbon. Both the anti-Sp77 and anti-Sp83 antibodies detected the nine outer doublets and the basal bodies of sea urchin sperm by immunofluorescence microscopy. In addition, the anti-Sp83 antibody, but not the anti-Sp77 antibody, detected a single 83 kDa polypeptide on immunoblots of unfertilized sea urchin egg cytoplasm, and a single polypeptide of 80 kDa on blots of isolated mitotic spindles from Chinese hamster ovary (CHO) cells. Previous studies have shown that tektins are present in the basal bodies and centrosomes/centrioles of cells ranging from clam to human. We found that anti-Sp83 decorates the spindle poles in sea urchin zygotes, and the interphase centrosome and spindle poles in CHO cells. In CHO cells arrested in S phase with aphidicolin, anti-Sp83 detects multiple centrosomes. The staining of the centrosome was not disrupted by prolonged nocodazole treatment, suggesting that the 80 kDa polypeptide is associated with the centrioles themselves. Our observations demonstrate that, like tektins, Sp77 and Sp83 are structural proteins associated with stable doublet microtubules, and may be components of basal bodies and centrioles of sea urchins and mammalian cells.
轴丝微管的生化分级分离产生原丝带（pf-带），这是一种由3 - 4条纵向原丝组成的不溶性结构，主要由α/β微管蛋白、轴丝蛋白A、B和C以及两种以前未鉴定的77 kDa和83 kDa的多肽组成。我们从紫海胆强壮柱头虫的精子鞭毛中分离出了77/83 kDa的多肽（称为Sp77和Sp83），并制备了针对它们的多克隆抗体。Sp77和Sp83仅与pf-带共纯化。抗Sp77和抗Sp83抗体通过免疫荧光显微镜检测到海胆精子的九个外双联体和基体。此外，抗Sp83抗体而非抗Sp77抗体在未受精卵细胞质的免疫印迹上检测到一条单一的83 kDa多肽，在中国仓鼠卵巢（CHO）细胞分离的有丝分裂纺锤体印迹上检测到一条80 kDa的单一多肽。先前的研究表明，轴丝蛋白存在于从蛤到人的各种细胞的基体和中心体/中心粒中。我们发现抗Sp83可标记海胆受精卵中的纺锤体极，以及CHO细胞中的间期中心体和纺锤体极。在用阿非迪霉素使CHO细胞停滞在S期时，抗Sp83可检测到多个中心体。中心体的染色不会因长时间用诺考达唑处理而受到干扰，这表明80 kDa的多肽与中心粒本身相关。我们的观察结果表明，与轴丝蛋白一样，Sp77和Sp83是与稳定双联体微管相关的结构蛋白，可能是海胆和哺乳动物细胞基体和中心粒的组成成分。
